Structure of the human neutrophil elastase gene

The gene for human neutrophil elastase (NE), a powerful serine protease carried by blood neutrophils and capable of destroying most connective tissue proteins, was cloned from a genomic DNA library of a normal individual. The NE gene consists of 5 exons and 4 introns included in a single copy 4-kilobase segment of chromosome 11 at q14. The coding exons of the NE gene predict a primary translation product of 267 residues including a 29-residue N-terminal precursor peptide and a 20-residue C-terminal precursor peptide. Analysis of the N-terminal peptide sequence suggests it contains a 27-residue "pre" signal peptide followed by a "proN" dipeptide, similar to that of other blood cell lysosomal proteases. The sequences for the mature 218-residue NE protein are included in exons II-V. The 5'-flanking region of the gene includes typical TATA, CAAT, and GC sequences within 61 base pairs (bp) of the cap site. The sequence 1.5 kilobases 5' to exon I contains several interesting repetitive sequences including six tandem repeats of unique 52- or 53-bp sequences. The 5'-flanking region also contains a 19-bp segment with 90% homology to a segment of the 5'-flanking region of the human myeloperoxidase (MPO) gene, a gene also expressed in bone marrow precursor cells and a protein stored in the same neutrophil granules as NE. In addition, like the MPO gene, the NE 5'-flanking region has several regions with greater than or equal to 75% homology to sequences 5' to c-myc, but there is no overlap between the NE-c-myc and MPO-c-myc homologous sequences.     

Identification of the sites on rabbit skeletal muscle protein phosphatase inhibitor-2 phosphorylated by casein kinase-II

Inhibitor-2 was phosphorylated by casein kinase-II in vitro at a rate similar to that of glycogen synthase, a physiological substrate of this protein kinase. The major phosphorylation sites were identified as serines-86, -120 and -121, the peptide containing serines-120 and -121 being labelled about 2.5-fold more rapidly than that containing serine-86. The 13 residues C-terminal to serine-121 (SGEEDSDLSPEERE) contain seven acidic amino acids, while the six residues following serine-86 (SDTETTE) contain three. These results are consistent with the known specificity requirements of casein kinase-II. The three serines are C-terminal to the threonine (residue 72) whose phosphorylation by glycogen synthase kinase-3 is potentiated by prior phosphorylation with casein kinase-II. This reinforces the view that a C-terminal phosphoserine residue is important for the specificity of glycogen synthase kinase-3. Identification of the residues phosphorylated by casein kinase-II will facilitate further studies on the in vivo phosphorylation state of inhibitor-2.     

Collagen fibrillogenesis in vitro: an investigation of the thermal memory effect and of the early events occurring during fibril assembly using dynamic light scattering

Dynamic light scattering has been used to characterize a variety of lathyritic rat skin collagen solutions. The technique was used to monitor the onset of fibril assembly in vitro and to investigate the thermal memory effect. Although the incorporation of thermal memory was demonstrated by reheating the sample and subsequently observing a shortened turbidimetric lag phase, no significant differences between naive solutions and ones exhibiting thermal memory could be detected using photon correlation spectroscopy. This suggests that subtle changes in the state of the collagen molecules rather than extensive changes in the degree of aggregation are responsible for the thermal memory effect. During fibrillogenesis, no large-scale changes in the distribution of monomers or aggregates occur until near the end of the lag phase.     

The protein phosphatases involved in cellular regulation. Primary structure of inhibitor-2 from rabbit skeletal muscle

The complete primary structure of inhibitor-2, a specific inhibitor of protein phosphatase-1, has been determined. The protein consists of a single polypeptide chain of 203 residues, and has a relative molecular mass of 22835 Da. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The threonyl residue phosphorylated by glycogen synthase kinase-3 is located at position 72. The molecule is very hydrophilic, lacks cysteine residues and the single tryptophanyl and phenylalanyl residues are at positions 46 and 139, respectively. The N-terminal alanyl residue is N-acetylated. Digestion with Staphylococcus aureus V8 proteinase, trypsin, or cleavage with cyanogen bromide, destroyed the biological activity of inhibitor-2, demonstrating that many large fragments (e.g. 1-49, 49-92, 67-101, 108-134, 142-182 and 163-197) are inactive. Digestion with clostripain generated a peptide comprising residues 25-114 which retained 2% of the inhibitory potency of the parent molecule. There is no sequence homology between inhibitor-2 and inhibitor-1.     

Vaginal estrus in unmated Belding's ground squirrels

Belding's ground squirrels are seasonally breeding rodents that have a single annual mating season (ca. 3 weeks long) which begins shortly after their vernal emergence from a 7-month period of hibernation. In this study, changes in vaginal estrus were assessed among unmated captive females. Following a 7-month period in a coldroom, vaginal lavages were taken daily to monitor changes in estrous condition. Females were in vaginal estrus within 24-48 hr of removal from the coldroom. Rather than exhibiting repeated cycles, adults (greater than or equal to 2 years old) remained in prolonged estrus (typically 3-4 weeks, but 8-10 weeks in some cases), whereas yearlings exhibited similar but shorter and possibly periodic changes in vaginal condition. The difference between the two age classes persisted in a second year of testing, indicating that the preadult status of yearlings (in the first year of testing) did not primarily account for the difference. In another experiment, removal from the coldroom was delayed for 24 days relative to adults removed at a time coincident with emergence from hibernation of free-living females. The "delayed" adults showed persistent vaginal estrus for a shorter total duration, such that both groups reached anestrus at approximately the same time. This implies that the latency to anestrus is not simply a fixed period from the time of removal from the coldroom.     

Serologic methods for detection of Pasteurella multocida infections in nasal culture negative rabbits

An agar gel-diffusion test (AGDT) and an enzyme-linked immunosorbent assay (ELISA) were utilized to detect serum antibodies against Pasteurella multocida in naturally infected rabbits derived originally from a Pasteurella-free colony. The antigen used in both assays was purified from a serotype 3 (P-1059) strain of P. multocida. Among 47 serum samples tested 15 (32%) were seropositive; 12 (26%) of which were both AGDT and ELISA-positive, while 3 (6%) were ELISA-positive only. All rabbits examined were normal clinically and negative to repeated nasal cultures, but subsequent cultures at necropsy demonstrated the presence of P. multocida in 11 of the AGDT-positive rabbits and in 14 of the ELISA-positive rabbits. The organism was isolated most frequently from the naso-oropharynx and the tympanic bullae. Serotyping of isolates recovered from the nasopharynx were determined to be serotype 3 or 3,12. Ten seronegative rabbits also were necropsied and none were found harboring P. multocida. These preliminary data indicate that the application of an enzyme-linked immunosorbent assay may prove efficacious in identifying apparently healthy, consistently nasal culture-negative rabbits as subclinical carriers of P. multocida.     

Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins

Summary Spinach cDNA libraries, made from polyadenylated seedling RNA, have been constructed in pBR322 and the expression vector gt11. Recombinant plasmids or phage for 14 intrinsic and peripheral thylakoid membrane proteins and one stromal protein have been identified. They encode components containing antigenic determinants against the lysine-rich 34 kd, the 23 kd and 16 kd proteins all associated with the water-splitting apparatus of the photosystem II reaction center, the ATP synthase subunits gamma, delta and CF_o-II, the Rieske Fe/S protein of the cytochrome b/f complex, subunits 2, 3, 5 and 6 of the photosystem I reaction center, plastocyanin, ferredoxin oxidoreductase, chlorophyll a/b-binding apoproteins of the lightharvesting complex associated with photosystem II, and the small subunit of the stromal enzyme ribulose bisphosphate corboxylase/oxygenase. The cDNA inserts lack complementarity to plastid DNA but hybridize to restricted nuclear DNA as well as to discrete poly A⁺-mRNA species. The precursor products obtained after translation of hybrid selected RNA fractions in a wheat germ assay are imported and processed by isolated unbroken spinach chloroplasts. The imported components comigrate with the respective authentic proteins.     

Photocontrol of Dark Circadian Rhythms in Stomata of Phaseolus vulgaris L

Stomatal diffusion resistance in primary leaves of Phaseolus vulgaris L. which had been grown in light:dark cycles followed a marked circadian rhythm when the plants were transferred to continuous darkness. Reentrainment of the rhythm required more than one inductive change in photoperiod. The phasing of the rhythm of dark stomatal opening was contolled primarily by the light-on (dawn) signal, whereas the rhythm of dark closure was related to the light-off (dusk) signal. The evidence points to a dual control of the circadian clock in which a product of photosynthesis plays a major role. No evidence for phytochrome involvement in the phasing of the rhythm was found. An influence of phytochrome on the amplitude of the stomatal rhythm was observed in which removal of phytochrome-far-red absorbing form caused rapid damping.     

Cloning of genes that encode a new heat-labile enterotoxin of Escherichia coli.

The genes for a new enterotoxin were cloned from Escherichia coli SA53. The new toxin was heat labile and activated adenylate cyclase but was not neutralized by antisera against cholera toxin or E. coli heat-labile enterotoxin. Subcloning and minicell experiments indicated that the toxin is composed of two polypeptide subunits that are encoded by two genes. The two toxin subunits exhibited mobilities on polyacrylamide gels that are similar to those of cholera toxin and E. coli heat-labile enterotoxin subunits. A 0.8-kilobase DNA probe for the new enterotoxin failed to hybridize with the cloned structural genes for E. coli heat-labile enterotoxin.     

Liver cytosolic aldehyde dehydrogenases from "alcohol-drinking" and "alcohol-avoiding" mouse strains: purification and molecular properties

Liver cytosolic aldehyde dehydrogenases (AHD-2) have been isolated in a highly purified state from "alcohol-drinking" (C57BL/6J) and "alcohol-avoiding" (DBA/2J) strains of mice. The purified enzymes were resolved into three major and one minor form of activity by isoelectric focusing (IEF) techniques and showed similar zymogram patterns. The enzymes had identical subunit sizes on SDS-polyacrylamide gels: 53,000. Gel exclusion chromatography, using Ultrogel AcA34, indicated that the enzymes were dimers. The enzymes exhibited biphasic kinetic characteristics and were readily distinguished from each other. The purified forms of AHD-2 from C57BL/6J and DBA/2J mice exhibited two apparent Km values in each case: 10 microM/100 microM and 30 microM/330 microM respectively. AHD-2 exhibited a broad pH optimum in the range 7.0-9.0 and was very sensitive towards disulphuram inhibition, with 50% inhibition occurring at 0.17 microM. The kinetic results support proposals that AHD-2 may be the primary enzyme for oxidizing acetaldehyde during ethanol oxidation in vivo.